Our current hypothesis is that mitochondrial trafficking and distribution is regulated in order to sense, integrate, and respond to changes in metabolic and growth status, synaptic activity, and pathological stress. Specific Aims 1-3 are formulated to address three fundamental questions: (Aim 1) how axonal mitochondria are recruited to and captured at active synapses; (Aim 2) how SNPH turns its anchoring on or off by sensing local ATP levels, and how the energy signaling enables neurons distribute axonal mitochondria into areas where energy consumption is high during development and regeneration; and (Aim 3) how neurons maintain and recover stressed mitochondria prior to the activation of Parkin-mediated mitophagy under physiological and pathological conditions. Specific Aim 1. Elucidate mechanisms anchoring mitochondria at presynaptic terminals. Presynaptic activity imposes large energetic demands that are met by local ATP synthesis via glycolysis and oxidative phosphorylation. We propose that mitochondrial anchoring at presynaptic terminals is regulated to sense synaptic activity and is required to sustain synaptic transmission. Our previous study revealed SNPH as a static anchor that holds axonal mitochondria stationary via microtubule interactions (Kang et al., Cell 2008). Deleting snph dramatically increases axonal mitochondrial motility in vitro and in vivo. Using the snph knockout (KO) mouse model, we further revealed an engine-brake switch mechanism, by which KIF5-SNPH interaction is regulated in order to move or stop axonal mitochondria in response to synaptic activity (Chen and Sheng JCB 2013). We also provided mechanistic insights into how motile axonal mitochondria contribute to the pulse-to-pulse variability of presynaptic strength, the most notable feature of synaptic transmission in response to repeated stimulations (Sun et al., Cell Reports 2013). ATP production from presynaptic mitochondria is the main energy source to sustain synaptic transmission. However, the mechanisms anchoring mitochondria at presynaptic terminals remain elusive. There is a critical need to understand how mitochondria are recruited to and retained at active synapses. We are testing our hypothesis that mobile axonal mitochondria can be captured at presynaptic terminals via SNPH-mediated anchoring to presynaptic actin filaments. This anchoring may ensure mitochondrial docking in response to change in synaptic activity and energy requirement by triggering energy sensing pathway to turn on and off the SNPH-actin anchoring mechanism. Specific Aim 2. Elucidate mechanisms linking energy sensing and mitochondrial transport to support neuronal growth and regeneration. Mitochondria trafficking to and anchoring at metabolically active regions provides local energy stations that constantly supply ATP. Regulation of mitochondrial transport and distribution is therefore a central issue concerning the maintenance of energy homeostasis throughout nerve cells. Neuronal growth and regrowth require high energy consumption to drive the synthesis of raw building materials and deliver these materials to growing tips. Proper mitochondrial transport into growth cones and injured axons ensures adequate ATP supply. Recent studies have established a correlation between polarized mitochondrial transport and axonal and dendritic morphology, thus neurons may have a unique mechanism for delivering mitochondria to distal axons by sensing energy requirements. AMPK serves as a master regulator of cellular energy homeostasis as it becomes activated when intracellular ATP supplies become depleted. We are testing our hypothesis that SNPH turns on and off its mitochondrial anchoring by sensing local ATP/ADP ratio through phosphorylation and dephosphorylation of SNPH, thus enabling neurons to re-mobilize and re-distribute axonal mitochondria into areas where energy consumption is in high demand. Specific Aim 3. Elucidate mechanisms maintaining and recovering stressed mitochondria. Parkin-mediated mitophagy is a key cellular pathway to eliminate damaged mitochondria in many non-neuronal cells. Our previous studies revealed that Parkin-mediated mitophagy is observed only in a small portion of mature neurons and occurs much more slowly than in non-neuronal cells (Cai et al., Current Biology 2012; Lin et al., Neuron 2017). In Parkin and Pink mutant flies, density and integrity of axonal mitochondria in motor neurons is comparable to WT. These findings argue for unique mechanisms that maintain and recover neuronal mitochondrial integrity and thus energy homeostasis in the early stages of mitochondrial stress, rather than acute global elimination of stressed mitochondria by activating Parkin-mediated mitophagy. In order to support this assumption, we are addressing two fundamental questions: (1) Can neurons recover chronically stressed mitochondria before Parkin-mediated mitophagy is activated? (2) Is mitophagy the last resort for mitochondrial quality control after recovery mechanisms have failed? Addressing these two questions is relevant to several major neurodegenerative diseases that associate with chronic mitochondrial stress. Our working model is that neurons have an intrinsic checkpoint mechanism for recovering stressed mitochondria through regulation of mitochondrial dynamics and ER-Mito contacts. If this pathway fails, Parkin-mediated mitophagy is subsequently activated to degrade stressed mitochondria. We propose that mitochondria-resident Mul1-Mfn2 pathway is critical to recovering stressed mitochondria, thus limiting neuronal mitophagy and maintaining energy homeostasis under chronic stress conditions. Publications: Kang, J.-S., J.-H. Tian, P. Zald, P.-Y. Pan, C. Li, C. Deng, and Z.-H. Sheng (2008). Docking of axonal mitochondria by syntaphilin controls their mobility and affects short-term facilitation. Cell 132, 137-148. Cai, Q., H. M. Zakaria, A. Simone, and Z.-H. Sheng (2012). Spatial parkin translocation and degradation of depolarized mitochondria via mitophagy in live cortical neurons. Current Biology 22, 545-552. Chen, Y. and Z.-H. Sheng (2013). Kinesin1-syntaphilin coupling mediates activity-dependent regulation of axonal mitochondrial transport. Journal of Cell Biology 202, 351-364. Sun, T., H. Qiao, P.-Y. Pan, Y. Chen, and Z.-H. Sheng (2013). Mobile axonal mitochondria contribute to the variability of presynaptic strength. Cell Reports 4, 413-419. Yuxiang Xie, Bing Zhou, Mei-Yao Lin, Shiwei Wang, Kevin D. Foust, and Zu-Hang Sheng. (2015) Endolysosome deficits augment mitochondria pathology in spinal motor neurons of asymptomatic fALS-linked mice. Neuron 87, 355-370. Morsci, N. S., D. H. Hall, M. Driscoll, and Z.-H. Sheng (2016). Age-related phasic patterns of mitochondrial maintenance in adult C. elegans neurons. Journal of Neuroscience 36, 1373-1385. Zhou, B., P. Yu, MY. Lin, T. Sun, Y. Chen, and Z.-H. Sheng (2016). Facilitation of axon regeneration by enhancing mitochondrial transport and rescuing energy deficits. Journal of Cell Biology 214, 203-119. Lin*, M-Y., X-T. Cheng* (equal contributions), P Tammineni, Y. Xie, B. Zhou, Q. Cai, and Z-H. Sheng (2017). Releasing syntaphilin removes stressed mitochondria from axons independent of mitophagy under pathophysiological conditions. Neuron 94, 595-610. Lin* M-Y, Cheng* X-T, Xie Y, Cai Q & Sheng Z-H (2017). Removing dysfunctional mitochondria from axons independent of mitophagy under pathophysiological conditions. Autophagy 13, 1792-1794. Sheng Z-H (2017). The interplay of axonal energy homeostasis and mitochondrial trafficking and anchoring. Trends in Cell Biology 27, 403-416 (Invited review).